1. Field of Invention
This invention relates to three dimensional nanofibrous scaffolds made from complex extracellular matrix (ECM) for tissue engineering applications.
2. Description of Related Art
Tissue engineering is a multidisciplinary field that involves the development of biological substitutes that restore, maintain or improve tissue functions. This field has the potential of overcoming the limitations of conventional treatments by producing a supply of organ and tissue substitutes biologically tailored to a patient. There is a continuing need in biomedical sciences for scaffolds of biocompatible compositions and of nanofibrous structure which closely mimic the composition and structure of natural ECM and which can be used in manufacturing devices for implantation within or upon the body of an organism.
Several techniques have been developed to produce tissue engineering scaffolds from biodegradable and bioresorbable polymers. For synthetic polymers, these are usually based on solvent casting-particulate leaching, phase separation, gas foaming and fibre meshes. For natural collagen scaffolds, these can be made by freezing a dispersion/solution of collagen and then freeze-drying it. Freezing the dispersion/solution results in the production of ice crystals that grow and force the collagen into the interstitial spaces, thus aggregating the collagen. The ice crystals are removed by freeze-drying which involves inducing the sublimation of the ice and this gives rise to pore formation; therefore the water passes from a solid phase directly to a gaseous phase and eliminates any surface tension forces that can collapse the delicate porous structure.
A major challenge for tissue engineering is to generate scaffolds which are sufficiently complex in mimicking the functions of the extracellular matrix (ECM) and yet not immunogenic. Different approaches to making tissue scaffolds have been described by Radisic et al., High-density seeding of myocyte cells for cardiac tissue engineering. Biotechnol Bioeng. 2003 May 20; 82(4):403-14; Boland et al., Electrospinning collagen and elastin: preliminary vascular tissue engineering. Front Biosci. 2004 May 1; 9:1422-32; Matthews et al., Electrospinning of collagen nanofibers. Biomacromolecules. 2002 March-April; 3(2):232-8; Huang et al., Engineered collagen-PEO nanofibers and fabrics. J Biomater Sci Polym Ed. 2001; 12(9):979-93.
However, complex ECM extracts, such as MATRIGEL and others, such as DECM from the submucosa of porcine small intestine (SIS, Hodde J P, Record R D, Liang H A, Badylak S F. Vascular endothelial growth factor in porcine-derived extracellular matrix. Endothelium. 2001; 8(1):11-24) or from the cornea (Desgranges P, Tardieu M, Loisance D, Barritault D. Extracellular matrix covered biomaterials for human endothelial cell growth (see Int J Artif Organs. 1992 December; 15(12):722-6) contain numerous differentiative cues which are not present in synthetic scaffolds or lost through conventional scaffold preparation techniques.
Other sources of ECM known to be effective for tissue remodeling include but are not limited to small intestine submucosa, stomach, bladder, alimentary, respiratory, or genital submucosa, or liver basement membrane. See, e.g., U.S. Pat. Nos. 6,379,710, 6,171,344; 6,099,567; and 5,554,389.
U.S. Pat. No. 5,939,323 to Valentini et al. describes hyaluronic acid derivitized scaffolds made by lyophilization. It was found that freeze/drying and air drying techniques did not yield interconnecting pores while lyophilizing from the wet state yielded scaffolds with interconnecting pores.
U.S. Pat. No. 6,787,357 to Bowlin et al. and US2004/022933A1 to Bowlin et al. disclosed the use of fibrin as ECM in forming an engineered tissue by electrospinning.
U.S. Patent Application Publication No. 2004/0191215A1 to Froix et al. disclosed compositions for initiating and promoting repair and regeneration of tissue.
U.S. Patent Application Publication No. 2004/0037813A1 to Simpson et al. described methods of making electroprocessed collagen and using the electroprocessed collagen in preparation of engineered tissue. Simpson et al. teach isolating collagen from tissue prior to electroprocessing or combining the isolated collagen with other proteins and substances to mimic ECM.
U.S. Pat. No. 6,398,819 to Bell described using animal tissues as starting materials for producing extracellular matrix particulates by a freeze drying process. The matrix particulates were then applied to collagen scaffolds, which can be seeded with living cells or the particulates may alone be seeded with living cells. This patent does not describe obtaining extracts of extracellular matrix or making scaffolds therefrom.
U.S. Patent Application Publication No. 20040166169A1 to Malaviya et al. described a method of making an implantable scaffold for repairing damaged or diseased tissue. The method includes the step of suspending, mixing, or otherwise placing pieces of a naturally occurring extracellular matrix material in a liquid. The naturally occurring extracellular matrix material and the liquid are formed into a mass. The liquid is subsequently driven off so as to form interstices in the mass. This patent does not describe obtaining extracts of extracellular matrix.
US2004/0258729A1 to Czernuszka et al. described a process for preparing a scaffold of polymer, generally a biocompatible polymer, ideally biodegradable or bioresorbable in nature for tissue engineering purposes, which comprises placing a composition comprising the polymer in mould possessing one or more voids therein, said mould being a negative of the desired shape of the scaffold, causing the polymer to acquire the shape of the mould, removing the mould and causing pores to be formed in the polymer, and without affecting the polymer. Czernuszka et al. do not describe making nanofibrous scaffolds from unfractionated ECM extracts.
A PCT Patent Application Publication No. WO05121316A by Bortolotto et al. disclosed a composition of matter useful in promoting cell growth including differentiation, proliferation, division and/or morphological changes in a cell or tissue, said composition comprising either a cell-based or cell-free extract of a muscle tissue preparation which preparation provides a source of, but not limited to, laminin, collagen I, collagen IV, entactin/nidogen, heparan sulfate proteoglycan as well as other components including cytokines and growth factors such as, but not limited to, one or more of EGF, bFGF, NGF, PDGF, IGF-1, TGF-B, VEGF and TNF-a or homologs thereof.
Attempts to make scaffolds suitable for tissue engineering applications mostly focus on mimicking the extracellular matrix by adding various individual components to the isolated collagen. It is known that intracellular matrix derived from different tissues varies in its composition and structure. Thus, reconstituting naturally occurring matrix is a formidable task
Basement membranes are thin, but continuous sheets that separate epithelium from stroma and surround nerves, muscle fibers, smooth muscle cells and fat cells. Basement membranes comprise type IV collagen, the glycoproteins laminin, entactin, nidogen and heparan sulfate proteoglycans. Various components of the basement membrane are known to interact with each other. In vitro studies with purified components show that laminin binds through its short chains to native but not to denatured type IV collagen and through a domain in its long chain to the heparan sulfate proteoglycan. Each of these basement membrane components is soluble. However, when these macromolecules are mixed together in vitro, they form a floccular precipitate containing laminin to type IV collagen to heparan sulfate proteoglycan in a 1:1:0.1 molar ratio. However, this precipitate lacks the resiliency and consistency expected of basement membranous structures. Purified components of basement membrane have been used previously as a coating for cultured cells. However, such material was soluble and did not form a three dimensional matrix.
U.S. Pat. No. 4,829,000 to Kleinman et al. disclosed making reconstituted basement membrane composition with biological activity capable of forming a three dimensional hydrogel matrix. The major components of the composition include laminin, type IV collagen, heparin sulfate proteoglycan, entactin and nidogen. It was shown that this composition can support cell adhesion, growth and differentiation beyond that known for the individual components. However, the three dimensional matrix described in this patent is a gel only. This patent does not describe making a fibrous structure or a scaffold mimicking the structure of extracellular matrix and therefore cannot be used as a scaffold for tissue engineering applications in its current form.
Therefore, despite the foregoing developments, there is a need in the art for fibrous or porous scaffolds for tissue engineering applications which can support growth and differentiation of cells.
All references cited in this disclosure are incorporated herein by reference in their entireties.